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This is an older age population, ranged from 25 to 71, with the mean around 46.

The gender was predominantly male. A lot of these males were working in a particular factory, a ceramics factory on night shift, and that was a work unit that probably was predominantly male and probably -- it may explain in part at least the gender predominance.

Twenty-two of the patients were known to have received antibiotic therapy after admission to the hospital for a mean of a little over 16 days, but you can see there's quite a bit of variation there.

The incubation period could be calculated for 30 patients and was a mean of 16 days. So that also is a long time. If you take the overall interval from exposure to death, it's got a very long range of six to 69 days. It's on the average about 20 days from the exposure time to death, but a wide range indicating that some of these organisms must remain as spores and germinate late, but the latest one was the germination period plus the incubation period, plus the disease period only added up to 69 days in this series.

The duration of illness is less than four days on the average, 3.85. Patients came to the hospital, and they didn't survive long. The average survival was just under a day.

Post mortem interval really did not correlate with any of the factors and probably indicates that autolysis is not an important factor in the analysis of the material.

Two observations that I haven't read about and need to examine the literature yet again that was impressive to us was the fact that there was a vascular damage, vasculitis and capillaritis, and these were identified in a high proportion of patients and believe they must be present in all of the patients.

And we believe that this is why the hemorrhages occur, that there's damage to blood vessels. It's not a very sophisticated idea, but I think that this is a very important component of the pathology of systemic anthrax.

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And this is an example of that vasculitis. Here we see a blood vessel with inflammation in the wall, fibrin rich edema adjacent to it.

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You see a very good example of necrosis of the blood vessel wall certainly weakened because of all the cells that are making up the media of this small vessel or necrotic.

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And here we see an example of an aneurism, a blood vessel here with a weakening of the wall and an out-pouching.



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Dr. Smith and Grinberg analyzed these materials with some semi-quantitative data in which they tried to quantify the number of organisms as they observed them histologically. If you'll notice that two-plus is somewhere between one and ten organisms per 25 objective field, to sort of see what a mid-range is.

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They also did a semi-quantitative tissue concentration of inflammatory cells, and there you can see that in the range of one would be up to ten cells per 25X objective field. So you're going to get some pretty high numbers before you really get into intense inflammation.

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Criteria for quantification of other parameters, and as is always true of pathology, it has the jeopardy of being subjective and not reproducible, but I believe that these actually are valid.



If you will notice that two, which they find as moderate and being present and significant; three marked prominent; and then down at one is really clearly present. So there's no question that it is there, but they don't believe that it was significant.

So they have put into their analysis, our analysis, a judgment factor where between one and two one shifts from it being there to it being there and being important.

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So as we look in the mediastinum and the peribronchial soft tissue in inhalational anthrax where most of the action really is, we find that there are, you know, 1.4 bacillus burden. So we're finding only about four organisms per 25X field, and then we found organisms in 54 percent of the cases.



You can see that the fibrin rich edema is more than two. So we're getting into a range of lesion in this location, and it's present in almost all of the tissues that were examined. So it is clearly a very important lesion in that location, as are the hemorrhages, both the permeating hemorrhages, the low pressure, and the high pressure hemorrhage.

You will notice that although you can find neutrophils among the nuclear cells, that the quantities are very low, and that those are -- there's not much inflammation occurring in response to this rapidly progressive infection.

An important lesion is lymphangitis. All of this process of increasing fluid exudation, transudation, and blockage of the lymphatics by all of the hemorrhage definitely is contributing to the accumulation of fluid in the thorax.

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So there is pneumonia, and I would like to try to clarify what I mean by pneumonia. When a pathologist says pneumonia, he really means just inflammatory consolidation of the lung. That does not necessarily mean that Bacillus anthracis germinated there, produced its toxin there, and caused the primary disease to be pneumonia, and indeed, I believe most of the lesions are caused by the bacilli, but they're probably caused by bacilli that are spreading back to the lung through the blood stream because we find most of the organisms in the blood.

Nevertheless, you can see some examples where the damage in the lung is localized.

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Another example, and you can see it's hemorrhagic, and hemorrhage is a theme I've probably already emphasized enough, and it also occurs in the lungs. So a lot of those consolidations are, indeed, hemorrhages.



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Some of the hemorrhage is tracking back along the bronchi from the mediastinum. So this is high pressure hemorrhage going back along the bronchi.

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And here we see an example of that high pressure hemorrhage that's displacing lung tissue, and that would give a consolidation, and you probably would prefer to think of that as hemorrhage than pneumonia.



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Here's a low pressure hemorrhage in which it's not distorted, but it is filling up the alveolar spaces with erythrocytes.

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Another area you get a true exudate. This is that fibrin rich edema occurring in the lung. Again, the pathology of anthrax can occur in the lung, as well as in other organs.



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And here is an example of what most people would truly think of as a bronchial pneumonia with exudate in the center, edema, and hemorrhage around the outside.

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There were seven of these patients who were arc welders, and there's evidence in those patients of pneumoconiosis. So you can see some scarring and deposition of hemosiderin in the lung associated with their profession.

We hypothesize that this scarring may have led to decreased clearance of organisms from the lung and caused some, in some cases, the possibility of the organism germinating in the lung because of its not being cleared efficiently, as efficiently to the mediastinal lymph nodes.

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To illustrate to you the lymphatic dilatation, this is a hugely dilated lymphatic vessel.

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Here's in the lung. We can see a pulmonary vessel here, and around the vessel these hugely dilated lymphatics. So this is -- there could be some fluid coming from the lung, but there may also be fluid that's backing up because of the damage in the mediastinum.

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Organisms can be found in these lymphatics with concentration being the greatest the closer one is to the mediastinum, decreasing as one goes into the lungs, but sometimes in association with an inflammatory lesion that one could call a lymphangitis.

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So the quantitative microscopic findings in the lungs are that we actually found bacilli in half of the lungs, but more than half of the bacilli were intravascular, indicating they're being spread to the lungs through the blood stream, and the patient has got systemic bacteremia.

Only about, you know, a sixth of them are intra-alveolar, and presumably those are spilling over from the blood vessels into the alveoli most of the time.

The exudates and neutrophils I think I've said enough about already. It's not an impressive inflammatory reaction, but there is some.

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There's also inflammation in the interstitium of a similar nature, and again, not very much of a cellular response.

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The hemorrhages, I think, are really very important, and a lot of things that we've been calling pneumonia really are due to the hemorrhage and due to congestion, the hemorrhage coming after the congestion, and due to damage to the capillaries, blood vessels, and with a lot of the increase of the mass due to lymphatic vessel obstruction, with dilatation being prominent.

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So pneumonia in the fatal cases of inhalational anthrax really probably is mostly due to hematogenous anthrax pneumonia, some due to retrograde lymphangetic pneumonia, possibly some pneumonia of anthrax organisms themselves, although I certainly cannot sort out an organism that's germinated in the lung in situ versus when it's spread to the lung in a germinated state.

Respiratory insufficiency is clearly a very important event, and it's greatly due to atelectasis, and I must not have emphasized it in the slide when we went over it, but the average volume of pleural effusions in these patients is over 1,700 milliliters. So that's like around 900 cc's on each side, and that's replacing lung tissue, compressing the lung.

You also have got the ascites in some patients elevating the diaphragm; got the expansion of the mediastinum because of the hemorrhage and because of the gelatinous edema, and so there's really a great deal of atelectasis, and I believe this is the primary or a major factor in patients' respiratory insufficiency.

There's really only one case we found of aspiration pneumonia, pulmonary edema per se or nosocomial pneumonia did not appear to be major factors that we identified.

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Hematogenous spread to the lung or to the brain we show so grossly causes this hemorrhage in the subarachnoid space. Here's the brain, and there's the meninges and the subarachnoid space full of blood.

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Here we can see it's a vasculitis, the blood vessel here, with inflammation in the wall and hemorrhage coming no doubt from hemorrhage from a damaged blood vessel.



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Bacilli very, very frequently found in the brain, in the subarachnoid space of the brain. Lots of Gram positive Bacillus anthracis.

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In a few cases there was hemorrhage in the brain parenchyma itself. We can see a blood vessel here with a ring hemorrhage. Again, the same phenomenon of vascular damages that I have emphasized enough.

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So the quantitative microscopic findings in the meninges were that we found organisms in 79 percent of the 29 brains that were taken at autopsy, and there was a significant amount of low pressure hemorrhage and fibrin, but the cellular response was modest.



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Hematogenous spread occurred also to the gastrointestinal tract. It's very well described, been in the Russian literature for well over five decades, and we see these high pressure hemorrhages in the submucosa. This is a small intestine that's been opened up, and we're looking at the luminal surface, and we see these localized areas of hemorrhage.

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Here we see histologically the mucosa here. It's lifted up by hemorrhage in the submucosa. This would be the submucosa, and a great big hematoma -- next slide -- will demonstrate how this comes about.



We see blood vessels in which the Gram positive bacilli are present, and so it's hematagomous spread to the intestine that results in these numerous lesions in the submucosa.

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So what are the mechanisms of death that we believe we have identified in inhalational anthrax? Atelectasis, as I've already discussed is a primary mechanism of death we felt in 39 of the cases and a major contributory mechanism of death in 46 percent of cases.

The hemorrhagic meningeal encephalitis is very important and is a primary mechanism of death in 34 percent of cases.

The pneumonia is -- and you've already gotten what I mean by pneumonia. It's all of those damages in the lung added up together, with the lung damage being the most important thing only in two out of the 41 cases.

Clearly there's more to the pathophysiology of systemic anthrax than one can see looking through a microscope, and the pathophysiology that Dr. Friedlander will tell you about will speak to that,b ut undoubtedly septic shock, which we attributed as being a contributing factor in 51 percent of the cases, could easily be more important than that, although these changes are pretty impressive, and I think that they really can kill you, that much pleural fluid accumulation and that severe hemorrhagic meningeal encephalitis.

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This is my last slide, and these patients were treated and admitted to the hospital, and some of them were treated, and some of them, they didn't treat them. I guess they didn't have in mind what the diagnosis was. They're rather non-specific symptoms, to begin with.

And so among the patients that were not treated, the organism was cultivated at autopsy from 83 percent, where those that received any treatment at all is received from only 23 percent.

The treatment that I have been told about is penicillin, cephalosporins, and forinphenocol (phonetic), and so I'm not sure of the doses, and certainly some of these patients didn't receive very much in the way of treatment in terms of duration.

In fact, those that were treated for less than 24 hours, we did continue to find organisms. Histologically we detected them in all of the patients that were not treated, and in 55 percent of those that were treated.

Histological detection of an organism, of course, doesn't prove that it's still alive. It could be that the dead organism is still lying there and has just not been removed yet.

Thank you. I'll be happy to answer any questions if I can.

CHAIRMAN RELLER: Are there questions from the panel for Dr. Walker?

Dr. Archer.

DR. ARCHER: Was there any evidence that antibiotic treatment had any effect on death? Of those who got antibiotics, was there a lower death rate than those who didn't?

DR. WALKER: Yes, some patients survived. I'm told that there were five survivors. There is the suggestion that there may have been prevention of death by some of the prophylaxis. Martin Hugh-Jones would be better to answer that, but it seems to be not the right curve of cases, and there's some missing towards the end that might have been exposed and got their disease prevented.

The five cases that survived, I have not had a chance to examine the records to know exactly ho long they had been ill and what they got treated with.

I think the image of anthrax as a virtually untreatable disease is probably close to true. I mean there are the experiments where they have taken animals and treated them with antibiotics past a certain critical phase, sterilized them of the organisms, but the animal still died of the effects of the toxin.

CHAIRMAN RELLER: Dr. Chesney.

DR. CHESNEY: Is the vasculitis present in all sizes of vessels? Is it in the larger vessels as well as the --

DR. WALKER: We didn't identify it in the larger vessels. We saw it mainly in medium sized and small vessels. I don't --

DR. CHESNEY: And is it your impression that the hemorrhage is coming from leakage from the medium and small sized vessels?

DR. WALKER: Yes.

CHAIRMAN RELLER: David, in Koch's day this was called splenic fever. What did the spleens look like in these patients?

DR. WALKER: We got a whole section in the article on that, on the spleen. I didn't think you guys would be interested in the spleen.

(Laughter.)

DR. WALKER: The spleen is enlarged, and it's got bacilli in it. No fatal lesions in the spleen though.

CHAIRMAN RELLER: Thanks.

Dr. Chikami.

DR. CHIKAMI: You describe in the pathology this high pressure hemorrhage within the mediastinum. Was there any evidence that this high pressure hemorrhage led to compression of other structures within the mediastinum and, say, compromise or led to something akin to cardiac tamponade or some effect on the cardiac function in these patients?

DR. WALKER: No.

CHAIRMAN RELLER: Thanks very much, Dr. Walker.

Oh, sorry. Dr. Friedlander.

DR. FRIEDLANDER: I just wanted to make a comment. First of all, it was a delight to see these pictures. I haven't seen them before.

And to make two points, if I might. One, to reemphasize this point that I think there's been some misconception, and I think people have tried to rectify it, that this is not primarily a pneumonia. This is a mediastinitis. This is a disease of the lymph node, and even these pulmonary findings appear to be mainly hemorrhagic findings in the lung.

The second relates to the question of survivors, and I was interested to hear that because as I read the articles -- and I think this is an important point, and I certainly don't have the answers for -- in the articles from Sverdlovsk, it said that there were nine survivors of inhalational disease.

As best I can read that data -- I wasn't there to visit either -- there is absolutely no evidence whatsoever given, either histologic, serologic, or microbiologic that these patients had anthrax.

So it becomes very difficult, I think, to posit, in fact, that there were survivors.

On the other hand, I think there is some data to suggest that in animals at least, in primates, that even when animals are bacteremic and even when they have mediastinitis, that some animals will survive.

So I don't think that it is -- at some point in any infection, there's a point of no return, but this concept, I think, is somewhat overstated that once bacteremia occurs or once mediastinitis occurs, it is absolutely fatal. I don't believe that myself, although I don't know any data in humans. There is some data in primates.

CHAIRMAN RELLER: Thank you.

And it's time for Dr. Friedlander to present the primate data.

DR. FRIEDLANDER: Thanks very much.

I appreciate Dr. Chikami and Meyerhoff asking me to present some of our previously reported work on post exposure prophylaxis in the non-human primate model of inhalational anthrax with antibiotics.

I'd like to begin with a few introductory remarks about the pathogenesis. I think we know a lot more; we've learned a lot more actually from Dr. Walker's presentation. We don't know a great deal about the disease in animals or in humans. What we know a great deal more about is the toxin and how it works in vitro, and that that relevance is to an in vivo situation remains still primarily conjectural.

Then I'll discuss some of the pathology in the non-human primate and contrast it to some extent with or compare it to that in the human, and finally present a review of the studies that we did during the Gulf War addressing the question of how to treat in the post exposure scenario.

Did that work? No, the lights. Okay.

You've heard this before, and I'll just reiterate it if I can find this thing. Okay. The spore is the infectious form that we're concerned about. Very rarely the bacillus can be infectious, but it's the spore particularly for inhalational disease.

It enters, as you heard, the skin, the GI tract or the lung. It is thought to germinate in the macrophage. This is the central player so far as we know either locally, if it's the skin or the GI tract, or is transported to a regional lymph node in the case of inhalational disease.

There there's the local production of toxins leading to edema and necrosis, the characteristic lesions that were pointed out just recently, and then spread from the node with bacteremia and toxemia.

This is shown -- this is an old slide from Dutz, who was a pathologist who studied this disease intensively or had a lot of experience with the disease, no nearly as much as in the Sverdlovsk now, and as I said, it's an old slide, but I like it because it points out this central player here.

Particularly for inhalational disease, this is the disease. It is not a pneumonia. It is a disease of the regional tracheal-bronchial, hiler (phonetic), mediastinal lymph node which spreads the mediastinum causing mediastinitis.

Now, the organism germinates -- you're going to do that for me. Okay.

This is a slide from Eli Metchnikoff. This disease as you heard is associated with the very origins of infectious disease and immunology. It was a big bacillus. It was easy to see under the microscope, and it was an important agricultural disease of domesticated animals.

The organism germinates. This is the macrophage from the liver of a rat. It germinates in -- the spore germinates in the macrophage.

Once the bacillus forms, it makes two toxins, edema toxin and lethal toxin that you've heard about. Both of them have anti-phagocytic effects. One of the prime manifestations of this disease in the cutaneous form and also in the inhalational form is a relative paucity of inflammatory cells. Malignant pustule is not a pustule, in fact. There are very few inflammatory cells.

So these two toxins probably have other effects as well, but we know in vitro they have dramatic effects on macrophages and neutrophils.

It also make a capsule, a polyglutamic acid capsule. Once that capsule is made and these organisms escape, they never see a phagocyte again. That capsule, as with many others, prevents phagocytosis. This is an extracellular infection once it is released from the macrophage.

Now, the characteristic finding, as we said, was when it's released was this spread to the lymph node, the damage to the lymph node, the hemorrhage, the necrosis, and then spread to the surrounding mediastinum.

From the mediastinum it spreads through the lymph, and you've seen dramatic pictures of lymphatic dilatation. It spreads from the lymph to the systemic circulation and to all the organs, particularly to the brain.

As we've heard and has been described before, about half of the cases have meningitis, and most often it's hemorrhagic.

Oh, sorry.

This is just a more modern version of Metchnikoff's slide. This happens to be an example of fluorescence microscopy from a group at the Pasteur Institute. These are mouse macrophages. You can barely see the outline of the cell, but it shows the spore essentially co-localized, both the F-actin into lysosomal markers.

So that the spores are ingested. They germinate. There's fusion, and eventually some of the spores germinate to the bacillus, destroy the cell, and the organism is now free to replicate.

Next slide, please.

Now, the clinical and pathologic findings of this disease were well described in the latter part of the 19th Century with the development of the industrial revolution. Basically a new disease appeared because for the first time people worked in an environment where there was a high concentration in an enclosed space of processed materials, wool, hair, hides.

That generated and produced a new disease that was recognized by the medical community. That was wood sorter's disease, or rag picker's disease in Austria and in Germany.

And this is from William Greenfield, whom you heard briefly about, and I just point out this statement, that great swelling of the bronchial glands occurred, these being sometimes completely broken down by hemorrhage and transformed into blood clots; extensive cellulitis together with hemorrhagic effusion around the bronchial glands and in the mediastinum generally.

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